Global Downregulation of Penicillin Resistance and Biofilm Formation by MRSA Is Associated with the Interaction between Kaempferol Rhamnosides and Quercetin

ABSTRACT The rapid development of methicillin-resistant Staphylococcus aureus (MRSA) drug resistance and the formation of biofilms seriously challenge the clinical application of classic antibiotics. Extracts of the traditional herb Chenopodium ambrosioides L. were found to have strong antibiofilm activity against MRSA, but their mechanism of action remains poorly understood. This study was designed to investigate the antibacterial and antibiofilm activities against MRSA of flavonoids identified from C. ambrosioides L. in combination with classic antibiotics, including ceftazidime, erythromycin, levofloxacin, penicillin G, and vancomycin. Liquid chromatography-mass spectrometry (LC-MS) was used to analyze the nonvolatile chemical compositions. Reverse transcription (RT)-PCR was used to investigate potential multitargets of flavonoids based on global transcriptional responses of virulence and antibiotic resistance. A synergistic antibacterial and biofilm-inhibiting activity of the alcoholic extract of the ear of C. ambrosioides L. in combination with penicillin G was observed against MRSA, which proved to be closely related to the interaction of the main components of kaempferol rhamnosides with quercetin. In regard to the mechanism, the increased sensitivity of MRSA to penicillin G was shown to be related to the downregulation of penicillinase with SarA as a potential drug target, while the antibiofilm activity was mainly related to downregulation of various virulence factors involved in the initial and mature stages of biofilm development, with SarA and/or σB as drug targets. This study provides a theoretical basis for further exploration of the medicinal activity of kaempferol rhamnosides and quercetin and their application in combination with penicillin G against MRSA biofilm infection. IMPORTANCE In this study, the synergistic antibacterial and antibiofilm effects of the traditional herb C. ambrosioides L. and the classic antibiotic penicillin G on MRSA provide a potential strategy to deal with the rapid development of MRSA antibiotic resistance. This study also provides a theoretical basis for further optimizing the combined effect of kaempferol rhamnosides, quercetin, and penicillin G and exploring anti-MRSA biofilm infection research with SarA and σB as drug targets.


INTRODUCTION
Staphylococcus aureus, the most common pathogen in human infection, can cause local purulent infection, pneumonia, pseudomembranous colitis, pericarditis, and even sepsis (1). Soon after penicillin was introduced for therapeutic use in 1940s, an increased incidence of penicillin resistance was reported in S. aureus strains (2).
Methicillin, a semi-synthetic penicillin, is resistant to penicillinase, and unfortunately, methicillin-resistant S. aureus (MRSA) emerged shortly after its clinical application (3). Today, MRSA is the leading cause of nosocomial infections (4). The prevalence of MRSA infection has resulted in the marginalization of the clinical use of traditional classic antibiotics including penicillin. On the other hand, the current development of new drugs is seriously lagging behind the rapid development of drug resistance, and the emergence of completely drug-resistant superbugs is bound to put patients in an incurable dilemma. Repurposing of old drugs with the idea of reducing toxicity is now considered as a potentially effective strategy to control MRSA infection (5,6).
Biofilms are microbial communities embedded in an extracellular matrix (ECM) composed of lipids, proteins, polysaccharides, and DNA that can form on medical implants or tissue surfaces to protect pathogenic bacteria from immune clearance and antibiotic killing, and are therefore implicated in most chronic infections (7,8).
Biofilms are formed in multi-stage process including initial attachment, accumulation, maturation and dispersion, and various virulence factors are involved in the progress along the stages of S. aureus biofilm formation (9). For example, surface protein adhesins, typically known as MSCRAMMs (microbial surface components recognising adhesive matrix molecules), including fibrinogen-binding proteins (FnBPs), fibrinogen-binding clumping factors (Clfs), elastin-binding protein (EbpS), and autolysin Atl, confer the ability for S. aureus to adhere to the host matrix (10).
Polysaccharide intercellular adhesin (PIA), also referred as poly-N-acetylglucosamine (PNAG) contributes to biofilm accumulation (11). Extracellular DNA (eDNA) released by bacterial autolysis can be an important component of the biofilm matrix (12). Proteases and phenol-soluble modulin (PSM) peptides may act as dispersants of biofilm to aid in bacterial dissemination and the settlement of new biofilms at a distant site (13). Moreover, the coordinated expressions of these virulence factors is modulated in complex networks that include the global regulators accessory gene regulator Agr, the staphylococcal accessory regulator SarA, and the alternative sigma factor B (σB) (14). Therefore, these virulence factors and their regulators are potential targets for effective prevention and control of S. aureus biofilm-associated infections.
Flavonoids are one of a large class of plant medicinal components with antioxidant, anti-inflammatory, anti-allergic, anti-cancer, anti-viral and anti-fungal properties (15). Recent studies have shown that, in addition to antibacterial effects, some flavonoids also inhibit bacterial biofilm formation by affecting bacterial adhesion, motility, and quorum sensing (QS) (16). Chenopodium ambrosioides L., an annual or perennial herb, is widely distributed on the planet and derived essential oil usually used in folk medicine as antirheumatic, anti-inflammatory, antipyretic, antihelmintic, antifungal, and anti-ulcer agents (17)(18)(19)(20)(21). However, the biological activities and mechanisms of action in terms of antibacterial and anti-biofilms remain poorly understood. In the previous stage, we compared the biofilm inhibitory activities of the products of alcohol extraction (AE) and water immersion extraction (IE) from root (GR), stem (GS), and ear (GE) of C. ambrosioides L. originated from Guangxi, China, and found that GE-AE had the most significant inhibitory effect on the biofilm formation by S. aureus ATCC43300. It was preliminarily estimated that flavonoids were the main active components. The purpose of this study was to investigate the antibacterial and anti-biofilm activities of flavonoids identified from C. ambrosioides L. in combination with traditional antibiotics including penicillin G against MRSA, and to investigate potential multi-targets of flavonoids based on global transcriptional responses of virulence and antibiotic resistance.

MATERIALS AND METHODS
Plant materials and strains. The C. ambrosioides L. plants were collected from Qinzhou prefecture in Guangxi, China. Fresh plant materials were separated into three parts including root (GR), stem (GS), and ear (GE) with leaves and seeds, dried in the shade after wash, then crushed, and stored at -20 ℃ prior to use. Three MRSA strains including standard strain of S. aureus ATCC43300, and two clinical strains of BWSA11 and BWSA15 isolated from burn wounds, were used in this study.
Preparation of C. ambrosioides L. extracts (CAEs). CAEs were prepared by using immersion extraction (IE) and alcohol extraction (AE), respectively. For IE, the dried and crushed plant samples were added with pure water at a solid-liquid ratio of 1:10 (g/v), extracted for 48 h. filtered by centrifugation, concentrated under reduced pressure, and freeze-dried to obtain GE-IE, GS-IE and GR-IE powders, respectively.
For AE, extracts of GE-AE, GS-AE and GR-AE were prepared by adding 50% ethanol to different plant parts at a ratio of 3:40 (g/v) with ultrasonic extraction for 30 min for two times. The extracts showing the highest anti-biofilm formation activity were further extracted using the fractional extraction with petroleum ether (PE), ethyl acetate (EA), and n-butanol (NB), respectively. All extracts including the residual aqueous layer (RA) were subjected to filtration, reduced pressure concentration, and freeze-drying. The obtained powders were dissolved in sterile water to achieve a high concentration reserve solution (40 mg/mL) prior to use.
Antibacterial activity assays. The antibacterial activities was determined by the disk diffusion or microdilution method according to the CLSI guidelines (22). In the disk diffusion test, the bacterial lawn of S. aureus ATCC43300 was prepared by evenly spreading 50 μL of bacterial cultures (approximately1.5×10 8 CFU/mL) on trypticase soy agar (TSA) with sterile cotton swabs, then the sterile filter paper discs (4 mm in diameter) impregnated with CAEs (200 μg/disc) were placed on agar surface. The diameter inhibition zone was determined after incubation for 20 h at 37°C. Kanamycin was used as a positive control.
For the microdilution test, serial twofold dilutions were made in concentrations ranging from 5 mg/mL to 0.3125 mg/mL for CAEs, and from 128 μg/mL to 4 μg/mL for antibiotics in sterile 96-well microplates. The initial populations of bacterial cells were approximately 5 × 10 5 CFU/mL. The minimum inhibitory concentration (MIC) of S. aureus strains of ATCC43300, BWSA11, and BWSA15 was determined at the lowest concentration of CAEs or antibiotics at which bacterial growth was reduced by more than 95% after incubation at 37 °C for 20 h. Hemolytic assays. A defibrated blood was prepared from a New Zealand rabbit prior to use. A TSB medium with bacteria inoculation approximately 1×10 6 CFU/mL in the absence or presence of CAE was incubated at 37°C for 20 h. The culture supernatant was prepared by centrifugation (5,000 × g) at 4°C for 5min, and followed by filtration using a Millipore membrane filter (0.22 μm). A mixture containing 200 μL bacterial supernatant, 775 μL 1 × PBS, and 25 μL defibrillated rabbit blood was incubated at 37°C for 30 min. A supernatant of the mixture was obtained by centrifugation at 5,000 × g for 1 min, and the absorbance was measured at 543 nm (A treatment ). A mixture without bacterial inoculation was used as negative control to reduce the background (A n-control ). A positive control (A p-control ) was obtained by mixing 975 μL of distilled water and 25 μL of defibrillated rabbit blood. The hemolytic activity of tested strain was expressed as a hemolysis ratio (%) = (A treatment - Polysaccharide intercellular adhesin (PIA) production assay. The production of PIA was evaluated by using Congo Red Agar method. Briefly, bacterial cells after spot inoculation on TSB agar with Congo red (50 μg/ml) in wells of 24-well plate were incubated at 37 °C for 24 hours. Black and brown lawns formed indicate that bacteria produced PIA, and red lawns formed indicate bacteria did not produce PIA.
Reverse Transcription-PCR assays. The transcriptional profile of S. aureus ATCC43300 in response to CAEs was assessed in this study. S. aureus ATCC4 3300 was chosen to evaluate the transcriptional changes of genes including icaA, agrA, rnaIII, sarA, luxS, fnbA, fnbB, clfA, clfB, ebpS, cidA, lrgA, sspA, psm, atlA, hld, vwb, and coa in response to CAEs. Briefly, the bacterial cells were cultivated in TSB in the absence or presence of GE-AE (2.5 mg/mL) at 37℃ for 24 h. After incubation, the bacterial cells were collected by centrifugation at 5,000 × g for 5 min, and then re-suspended with TE buffer. Total RNA was extracted using Trizol (Solarbio Bio. Inc., Beijing, China). The cDNA was synthesized using TianGen FastKing gDNA Dispelling RT SuperMix Kit according to the manufacturers' protocol. The PCR amplification was performed using the ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA) in a 20-μL reaction mixture containing 10 μL of 2 × QuantiTect SYBR green PCR master mix (Qiagen), 2 μL of cDNA, and 10 pmol/μL each primer ( Table 1). The 16s rRNA was used as the reference gene, and the comparative threshold cycle (∆C T ) method was used to analyze the relative expression of the targeted genes. Statistical analysis. Data collected were analyzed using SPSS 25 and GraphPad Prism 8. All data were presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to determine the differences between the test groups.

Quercetin optimized biofilm inhibition by kaempferol glycosides against S.
aureus. Significant inhibitory activity against biofilm formation was observed for all extracts of C. ambrosioides L. (Figure S1), but no significant inhibitory activity against the growth of S. aureus ATCC43300 ( Figure S2). It was observed that, among the 6 products of GS, GR and GE extracted by IE and AE, GE-AE exhibited relatively high biofilm inhibition activity against S. aureus ATCC43300, while mong the fractionated extracts of GE-AE, GE-AE-EA showed the highest biofilm inhibition activity ( Figure S1). In contrast, the biofilm inhibitory activity of GE-AE was significantly higher (P < 0.05) than that observed for GE-AE-EA at concentrations ranging from 0.3125 to 2.5 mg/ml ( Figure 1). This could be due to chemical changes in various kaempferol glycoside derivatives including kaempferol-3,7-dirhamnoside, Furthermore, GE-AE (> 62.5 μg/ml) was found to effectively inhibit biofilm formation by S. aureus BWSA11 (Figure 2), a clinical MRSA strain with robust biofilm-forming ability (28). The biofilm-inhibitory activity of GE-AE was further found to be significantly correlated with kaempferol-3,7-dirhamnoside, a representative kaempferol glycoside, but not with quercetin and rutin ( Table 2). This result is in agreement with a previous finding that kaempferol inhibited the primary attachment phase of biofilm formation in S. aureus (29). It was further found that quercetin (1.95~7.81 μg/ml) and kaempferol-3,7-dirhamnoside (15.63~125 μg/ml) had a synergistic inhibitory effect on S. aureus BWSA11 biofilm (Figure 3), while rutin and kaempferol-3,7-dirhamnoside did not (data not shown). This finding could explain the higher biofilm inhibitory activity ( Figure S1), but the lower total content of kaempferol glycosides in GE-AE (Table 1) (Figure 4). This observation was in agreement with the result that kaempferol had less bacteriostatic activity against S. aureus (31). Considering the extremely low content of quercetin in GE-AE, it is not difficult to explain why the antibacterial property of GE-AE was closely related to kaempferol glycosides (Table   3). Limited by the solubility of kaempferol-3,7-dirhamnoside, it is difficult to determine whether there was a synergistic or additive antibacterial effect between quercetin and kaempferol-3,7-dirhamnoside against the growth of S. aureus BWSA11 (FIC < 1.5). However, as shown in Figure 5, the presence of kaempferol-3,7-dirhamnoside (125 and 250 μg/ml) and quercetin (15.63 μg/ml) reduced each other'MIC value against S. aureus BWSA11 by 2-fold and at least 8-fold. Therefore, a proper compatibility of kaempferol glycosides and quercetin is beneficial to the mutual promotion of their antibacterial activities. Furthermore, GE-AE in combination with penicillin G was found to synergistically inhibit the growth of BWSA11, BWSA15, and ATCC43300 with FICs of < 0.19, < 0.5, and < 0.16, respectively ( Figure 5). Interestingly, neither the combination of quercetin and penicillin G nor the combination of kaempferol-3,7-dirhamnoside and penicillin G appeared to synergistically inhibit the growth of representative strains tested in this study (data not shown). This is in agreement with a previous finding that kaempferol and quercetin had mild inhibitory effects on β-lactamase when used alone, but exhibited excellent β-lactamase inhibition when used in combination with rifampicin (32). Therefore, the combined antibacterial effect of GE-AE with penicillin G was likely to depend on the interaction of kaempferol glycosides and quercetin with penicillin G. The combination of GE-AE with other antibiotics did not show a synergistic inhibitory activity against MRSA strains.
Moreover, GE-AE combined with penicillin G at various combinations tested significantly inhibited the biofilm formation by S. aureus BWSA11, BWSA15, and ATCC43300, respectively, and most of the combinations synergistically inhibited the biofilm formation by S. aureus BWSA15 and ATCC43300 ( Figure 6). The combination of GE-AE and penicillin G did not show a synergistic inhibitory effect on the biofilm of BWSA11, which may be attributed to the extremely sensitive nature of biofilm formation by S. aureus BWSA11 to GE-AE at all concentrations tested ( Figure 6). It was further observed that kaempferol-3,7-dirhamnoside (15.63~250 μg/ml) combined with penicillin G (16~128 μg/ml) also significantly or synergistically inhibited the biofilm formation by S. aureus BWSA11 ( Figure 6).
Unlike the combination of kaempferol-3,7-dirhamnoside and penicillin G, the combination of quercetin and penicillin G at certain concentrations instead induced the biofilm formation by S. aureus BWSA11 ( Figure S3). Therefore, the synergistic inhibitory effect of GE-AE and penicillin G on the biofilm formation of representative strains of S. aureus was also unlikely to be the result of the interaction between penicillin G and a single component.

Global down regulation of penicillin resistance and biofilm formation by S.
aureus in response to GE-AE was with agr, sarA, and sigB rather than luxS. A total of 17 genes functionally contributing to penicillin resistance and/or biofilm formation (Table S2) were selected to determine the transcriptional response of S. aureus. As shown in Figure 7, these contributors are transcriptionally interconnected to agr (33)(34)(35)(36), luxS (37)(38)(39), sarA (40)(41)(42), and sigB (43)(44)(45). Specifically for agr-dependent regulation of PSM, the dramatic effect of agr on psmα expression is mediated by the direct binding of the AgrA response regulator, which occurs independently of RNAIII, the small regulatory RNA (46). It was found that the expression of all these contributors and their potential regulators was down-regulated

GE-AE-induced down-regulation of penicillinase was main responsible for
increased susceptibility of MRSA to penicillin G. As summarized in Table 4, penicillin resistance is associated not only with the production of penicillinase (blaZ), but also with the presence of PBP2a (mecA), a transpeptidase enzyme that presents very low beta-lactam affinity. Furthermore, murein hydrolysis caused by up-regulation of CidA (cidA) (50), the positive regulator of autolysis Atl (atlA), or down-regulation of the negative regulator LrgA (lrgA) (51) may also be associated with penicillin resistance. In this study, contributors including blaZ, mecA, cidA, and atlA were found to be transcriptionally down-regulated in S. aureus ATCC43300 in the presence of GE-AE (0.31~2.5 mg/mL) (Figure 8). Inactivation of LuxS/AI-2 has also been reported to result in decreased autolysis and decreased susceptibility to cell wall synthesis inhibitor antibiotics such as penicillin, oxacillin, vancomycin, and teicoplanin (39,52). Although mecA expression was significantly down-regulated, the bacteriostatic activity of ceftazidime, a β-lactam antibiotic, as well as vancomycin against S. aureus ATCC43300 was contrarily found to be reduced (data not shown).
This observation was consistent with decreased expression of luxS, cidA and atlA, indicating a decreased cell wall hydrolytic activity of S. aureus ATCC43300 in response to GE-AE. Therefore, the increased susceptibility of S. aureus ATCC43300 to penicillin G was mainly due to reduced production of penicillinase in response to GE-AE. Given that the synchronous expression ( Figure 9) of agr (agrA and rnaIII) and blaZ contradicts their negative transcriptional relationship (Figure 7), it is likely that sarA was primarily responsible for the down-regulation of penicillinase.  (Table S), (Table 4), which mechanistically involves various aspects of biofilm formation. The multi-targeted biofilm inhibitory property of GE-AE was also corroborated by significant reductions in cell adhesion (Figure 9 and10), erythrocyte lysis (Figure 9, 11, and S4) and PIA production (Figure 9 and S5) in S. aureus ATCC43300 and clinical representatives. Certain flavonoids can effectively inhibit toxic expressions such as α-hemolysin (53). The down-regulation of hld, psmα, and sspA suggests that inhibition of S. aureus ATCC43300 biofilm by GE-AE was independent of dissociation activity. Furthermore, agr (35,41) was found to be associated with positive down-regulation of crtN, hla, and mecA, accounting for 25% of biofilm-positive contributors, while sigB and sarA were associated with 92% and 83% of biofilm-positive contributors, respectively (Table 4). Exceptionally, mecA (54) can in turn inhibit the activity of agr (Figure 7), thereby indirectly affecting the expression of related contributors. But judging from the synchronized expression of mecA and agr (Figure 9), the down-regulation of mecA was likely to be a concomitant phenomenon rather than a cause of the down-regulation of biofilm expression. It has been reported that extracellular DNA released from Atl-dependent autolysis is mainly responsible for the early stages of MRSA biofilm formation (55), whereas the FnBPs promote subsequent intercellular accumulation and biofilm maturation (56). This observation is consistent with the the down-regulation of atlA and fnbA and accompanied biofilm attenuation in S. aureus ATCC43300 in response to GE-AE.

GE-AE targeting
Taking into account the regulatory relationship among atlA, fnbA, sarA, and sigB, the down-regulation of atlA is most likely due to reduced expression of sigB, while the down-regulation of fnbA is likely to be related to both sigB and sarA.
In conclusion, the synergistic antibacterial and inhibitory biofilm activity of C. ambrosioides L. alcohol extract combined with penicillin G against MRSA was closely related to the interaction between main components of kaempferoside glycosides and quercetin. In mechanism, the increased sensitivity of MRSA to penicillin G was mainly related to the down-regulation of penicillinase expression with SarA as a potential drug target, while the biofilm inhibitory activity is mainly related to down regulation of various virulence factors involved in the initial and mature stages of biofilm development with SarA and/or σB as potential drug target.
This study provides a theoretical basis for further exploration of the medicinal activity of kaempferol rhamnosides and quercetin and its application in combination with the classic old drug penicillin G against MRSA biofilm infection.

TRANSPARENCY DECLARATIONS
None to declare.

SUPPLEMENTARY DATA
Supplementary data Table S1, Table S2, Table S3, Figure S1, Figure S2, Figure S3, Figure S4, and Figure S5 are available.           All data are presented as means of three biological replicates.      All data are presented as means of three biological replicates.

Responses to Reviewers
Re: Spectrum02782-22 (Global down-regulation of penicillin resistance and biofilm formation by MRSA is associated with the interaction between kaempferol rhamnosides and quercetin) Authors: Xinlong He, et al.
We sincerely thank all Reviewers for your valuable comments and giving us the chance to revise our manuscript. We have finished the point-by-point responses for this manuscript, and you will find the changes in the Marked Up Manuscript up-loaded.

Responses to Reviewer #1 (Comments for the Author):
This study is well designed and written based on the results obtained. There are few minor comments.
1. In Table 1, add the concentration of each compound with relative proportions.
Response: Due to the inaccessibility of some standards, we did not calculate the actual concentrations of these compounds. Instead, we performed qualitative analysis on these compounds identified by MS and relative quantitative analysis based on their LC abundances. Since we do not know the actual concentrations, to make it clear that two extracts are comparable, we annotate the results of the difference analysis in 2. State the reason why the penicillin was used rather than other generations of beta-lactams or other classes of antibiotics.

Dr. Feng Lu Yangzhou University Yangzhou China
Re: Spectrum02782-22R1 (Global down-regulation of penicillin resistance and biofilm formation by MRSA is associated with the interaction between kaempferol rhamnosides and quercetin) Dear Dr. Feng Lu: Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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